R-Loop Formation Is a Distinctive Characteristic of Unmethylated Human CpG Island Promoters

Ginno, Paul A.; Lott, Paul; Christensen, Holly C.; Korf, Ian F; Chédin, Frédéric

doi:10.1016/j.molcel.2012.01.017

Cited by 718 publications

(1,119 citation statements)

References 65 publications

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“…To test this model, we assayed R-loop formation genome-wide in SH-SY5Y cells expressing the control or SMN shRNA using DNA-RNA immunoprecipitation (IP) sequencing (DRIPseq), which enriches for hybrid structures in the chromatin using the R-loop-specific antibody S9.6 (44). We also performed DRIPseq on samples immunoprecipitated with mouse IgG as a control for specific enrichment.…”

Section: Smnmentioning

confidence: 99%

SMN deficiency in severe models of spinal muscular atrophy causes widespread intron retention and DNA damage

Jangi

Fleet

Cullen

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

107

111

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Spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disease, is the leading monogenic cause of infant mortality. Homozygous loss of the gene survival of motor neuron 1 (SMN1) causes the selective degeneration of lower motor neurons and subsequent atrophy of proximal skeletal muscles. The SMN1 protein product, survival of motor neuron (SMN), is ubiquitously expressed and is a key factor in the assembly of the core splicing machinery. The molecular mechanisms by which disruption of the broad functions of SMN leads to neurodegeneration remain unclear. We used an antisense oligonucleotide (ASO)-based inducible mouse model of SMA to investigate the SMN-specific transcriptome changes associated with neurodegeneration. We found evidence of widespread intron retention, particularly of minor U12 introns, in the spinal cord of mice 30 d after SMA induction, which was then rescued by a therapeutic ASO. Intron retention was concomitant with a strong induction of the p53 pathway and DNA damage response, manifesting as γ-H2A.X positivity in neurons of the spinal cord and brain. Widespread intron retention and markers of the DNA damage response were also observed with SMN depletion in human SH-SY5Y neuroblastoma cells and human induced pluripotent stem cell-derived motor neurons. We also found that retained introns, high in GC content, served as substrates for the formation of transcriptional R-loops. We propose that defects in intron removal in SMA promote DNA damage in part through the formation of RNA:DNA hybrid structures, leading to motor neuron death.

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Section: Smnmentioning

confidence: 99%

SMN deficiency in severe models of spinal muscular atrophy causes widespread intron retention and DNA damage

Jangi

Fleet

Cullen

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

107

111

View full text Add to dashboard Cite

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“…61,62 In contrast, R-loops play beneficial roles by mediating CRISPR interference, 63,64 promoting IgG class switching, 65,66 and by regulating transcription. 67,68 These structures can form in cis 60 or trans 69 in vivo, either by "threading back" of nascent RNAs during transcription by RNA Pol II, through the action of the homologous recombination machinery, or associated RNA-binding proteins as in the case of CRISPR. 60,70,71 Given that RNP assembly defects cause R-loops, 60,70 we speculated that loss of DBP2 might promote formation of these structures between the GAL lncRNAs and GAL gene promoters.…”

mentioning

confidence: 99%

LncRNAs: Bridging environmental sensing and gene expression

2016

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The survival of all organisms is dependent on complex, coordinated responses to environmental cues. Non-coding RNAs have been identified as major players in regulation of gene expression, with recent evidence supporting roles for long non-coding (lnc)RNAs in both transcriptional and post-transcriptional control. Evidence from our laboratory shows that lncRNAs have the ability to form hybridized structures called R-loops with specific DNA target sequences in S. cerevisiae, thereby modulating gene expression. In this Point of View, we provide an overview of the nature of lncRNA-mediated control of gene expression in the context of our studies using the GAL gene cluster as a model for controlling the timing of transcription.

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“…R-loops have been suggested to play a key role in preventing DNA methylation at CGI. 36 Our group has recently shown that GC skew additionally defines key points of RNAP II pausing in CGI promoters, 37 regulating its release into elongation. In previous work, we showed that the re-establishment and persistence of RNAP II, even in its paused state impedes remethylation and gene re-silencing after DAC-induced demethylation.…”

Section: Discussionmentioning

confidence: 99%

Factors affecting the persistence of drug-induced reprogramming of the cancer methylome

et al. 2016

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Aberrant DNA methylation is a critical feature of cancer. Epigenetic therapy seeks to reverse these changes to restore normal gene expression. DNA demethylating agents, including 5-aza-2 0 -deoxycytidine (DAC), are currently used to treat certain leukemias, and can sensitize solid tumors to chemotherapy and immunotherapy. However, it has been difficult to pin the clinical efficacy of these agents to specific demethylation events, and the factors that contribute to the durability of response remain largely unknown. Here we examined the genome-wide kinetics of DAC-induced DNA demethylation and subsequent remethylation after drug withdrawal in breast cancer cells. We find that CpGs differ in both their susceptibility to demethylation and propensity for remethylation after drug removal. DAC-induced demethylation was most apparent at CpGs with higher initial methylation levels and further from CpG islands. Once demethylated, such sites exhibited varied remethylation potentials. The most rapidly remethylating CpGs regained >75% of their starting methylation within a month of drug withdrawal. These sites had higher pretreatment methylation levels, were enriched in gene bodies, marked by H3K36me3, and tended to be methylated in normal breast cells. In contrast, a more resistant class of CpG sites failed to regain even 20% of their initial methylation after 3 months. These sites had lower pretreatment methylation levels, were within or near CpG islands, marked by H3K79me2 or H3K4me2/3, and were overrepresented in sites that become aberrantly hypermethylated in breast cancers. Thus, whereas DAC-induced demethylation affects both endogenous and aberrantly methylated sites, tumorspecific hypermethylation is more slowly regained, even as normal methylation promptly recovers. Taken together, these data suggest that the durability of DAC response is linked to its selective ability to stably reset at least a portion of the cancer methylome.

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R-Loop Formation Is a Distinctive Characteristic of Unmethylated Human CpG Island Promoters

Cited by 718 publications

References 65 publications

SMN deficiency in severe models of spinal muscular atrophy causes widespread intron retention and DNA damage

SMN deficiency in severe models of spinal muscular atrophy causes widespread intron retention and DNA damage

LncRNAs: Bridging environmental sensing and gene expression

Factors affecting the persistence of drug-induced reprogramming of the cancer methylome

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